This invention pertains to a series of new derivatives of 2,4,6-trimethyl-1,4-dihydropyridine-3,5-dicarboxylic acid, their synthesis, and the use of these compounds as platelet-activating factor antagonists, inhibitors of certain protein kinases, transcriptional inhibitors of COX-2 expression, and as agents effective in protecting brain tissue from injuries related to trauma or disease.
Various derivatives of 1,4 dihydropyridines have been described with properties that include one or more of the following: platelet activating factor antagonist activity, coronary vessel dilators, antihypertensives, antiischemic, antithrombotic activity, cerebral vessel dilators, peripheral vessel dilators, renal vessel dilators. See U.S. Pat. Nos. 5,177,211; 5,070,205; 5,068,337; 4,937,242; 4,801,598; 4,788,205; 4,755,512; 3,996,234; and 3,974,274. See also, WO 90/12015 and EPO 0325 187.
It has been known for sometime that brain ischemia promotes the accumulation of arachidonic acid. See N. G. Bazan, xe2x80x9cEffects of ischemia and electroconvulsive shock on free fatty acid pool in the brain,xe2x80x9d Biochim. Biophys. Acta, vol. 218, pp. 1-10 (1970); N. G. Bazan, xe2x80x9cChanges in free fatty acids of brain by drug induced convulsions, electroshock and anesthesia,xe2x80x9d J. Neurochem., vol. 18, pp. 1379-1385 (1971). In addition, newborn mammals and adult poikilotherms, unlike adult rodents and nonhuman primates, do not display an arachidonic acid accumulation induced by ischemia. See Bazan 1971; N. G. Bazan et al., xe2x80x9cRegional distribution and rate of production of free fatty acids in rat brain,xe2x80x9d J. Neurochem., vol. 18, pp.1387-1393 (1971); and M. I. Aveldano et al., xe2x80x9cDifferential lipid deacylation during brain ischemia in a homeotherm and a poikilotherm. Content and composition of free fatty acids and triacylglycerols,xe2x80x9d Brain Res., vol.100, pp. 99-110 (1975). A connection has been suggested between brain damage and both the accumulation of arachidonic acid and the activation of phospholipase A2, because resistance to brain damage was shown in animals that do not accumulate arachidonic acid (e.g., newborn mammals and mature poikilotherms) but not in animals that accumulate arachidonic acid (e.g., adult mammals). This pattern of resistance to brain damage was shown in animal models subjected to ischemia, stroke, cerebral edema, and epilepsy. See N. G. Bazan et al., xe2x80x9cMembrane lipids in the pathogenesis of brain edema: Phospholipids and arachidonic acid, the earliest membrane components changed at the onset of ischemia,xe2x80x9d In: Advances in Neurology, Vol 28: Brain Edema. (J. Cervxc3x3s-Navarro and R. Ferszt, eds), Raven Press, New York, pp 197-205 (1980); N. G. Bazan et al., xe2x80x9cEndogenous pools of arachidonic acid enriched membrane lipids in cryogenic brain edema,xe2x80x9d In: Recent Progress in the Study of Brain Edema, (K. G. Go and A. Baethmann, eds), Plenum Press, New York, pp 203-212 (1984); and N. G. Bazan et al., xe2x80x9cFree arachidonic acid and membrane lipids in the central nervous system during bicuculline induced status epilepticus,xe2x80x9d In: Advances in Neurology Vol 34: Status Epilepticus, (A. V. Delgado-Escueta, C. G. Wasterlain, D. M. Treiman, R. J. Porter, eds), Raven Press, New York, pp 305-310 (1983).
Phospholipase A2 generates the platelet-activating factor (xe2x80x9cPAFxe2x80x9d) precursor as well as arachidonic acid. Platelet-activating factor (PAF) accumulation has been shown to participate in ischemia-reperfusion brain damage and excitotoxic neuronal injury. See N. G. Bazan, xe2x80x9cInflammation: A signal terminator,xe2x80x9d Nature, vol. 374, pp.501-502 (1995); N. G. Bazan et al., xe2x80x9cPlatelet-activating factor is both a modulator of synaptic function and a mediator of cerebral injury and inflammation,xe2x80x9d In: Advances in Neurology, Vol. 71 :Cellular and Molecular Mechanisms of Ischemic Brain Damage, (B. Siesjxc3x6 and T. Wieloch, eds.), Lippincott-Raven Publishers, Philadelphia, vol. 37, pp. 475-484 (1996b); N. G. Bazan et al., xe2x80x9cPlatelet-activating factor and other bioactive lipids,xe2x80x9d In: Cerebrovascular Disease, Pathophysiology, Diagnosis and Management (M. D. Ginsberg and J. Bogousslavsky, eds.) Chapter 37, Blackwell Science Publishers, Malden, Mass., pp. 532-555 (1998); N. G. Bazan et al., xe2x80x9cMembrane-derived lipid second messengers as targets for neuroprotection: Platelet-activating factor,xe2x80x9d In: Emerging Strategies in Neuroprotection, Advances in Neuroprotection (P. J. Marangos and H. Lal, eds.), Birkhxc3xa4user, Boston, pp. 238-251 (1992); T. Panetta et al., xe2x80x9cEffects of a platelet-activating factor antagonist (BN 52021) on free fatty acids, diacylglycerols, polyphospho-inositides and blood now in the gerbil brain: Inhibition of ischemia reperfusion induced cerebral injury,xe2x80x9d Biochem. Biophys. Res. Comm., vol.149, pp.580-587 (1987); K. Nishida et al., xe2x80x9cPlatelet-activating factor in brain regions after transient ischemia in gerbils,xe2x80x9d Stroke, vol. 27, pp.514-519 (1996); S. A. L. Bennett et al, xe2x80x9cPlatelet-activating factor receptor expression is associated with neuronal apoptosis in an in vivo model of excitotoxicity,xe2x80x9d Cell Death Differentiation, vol. 5, pp. 867-875 (1998); H. Bito et al., xe2x80x9cPlatelet-activating factor (PAF) receptor in rat brain: PAF mobilizes intracellular Ca2+ in hippocampal neurons,xe2x80x9d Neuron, vol.9, pp. 285-294 (1992); B. Bonavida et al., xe2x80x9cPlatelet-activating factor and the cytokine network in inflammatory processes,xe2x80x9d Clin. Rev. Allergy, vol. 12, pp. 381-395 (1994); G. Feurstein et al., xe2x80x9cPlatelet-activating factor: a putative mediator in central nervous system injury?,xe2x80x9d Stroke, vol. 21(suppl III), pp. III-90-III-94 (1990); and H. A. Gelbard et al., xe2x80x9cPlatelet-activating factor: a candidate human immunodeficiency virus type 1-induced neurotoxin,xe2x80x9d J. Virol., vol. 68, pp. 4628-4635 (1994).
PAF, a potent phospholipid messenger, is released during ischemic insults to the brain and after seizures. See R. Kumar et al., xe2x80x9cProduction and effects of platelet-activating factor in the rat brain,xe2x80x9d Biochem. Biophys. Acta, vol. 963, pp.375-383 (1988). Multiple receptors and multiple functions for PAF has been reported. There are seven transmembrane-spanning domain receptors as well as intracellular binding sites for PAF. See Z. Honda et al., xe2x80x9cTransfected platelet-activating factor receptor activates mitogen-activated protein (MAP) kinase and MAP kinases kinases in Chinese hamster ovary cells,xe2x80x9d J. Biol. Chem., vol. 269, pp.2307-2315 (1994); V. L. Marcheselli et al., xe2x80x9cDistinct platelet-activating factor binding sites in synaptic endings and in intracellular membranes of rat cerebral cortex,xe2x80x9d J. Biol. Chem., vol. 265, pp. 9140-9145 (1990); and V. L. Marcheselli et al., xe2x80x9cPlatelet-activating factor is a messenger in the electroconvulsive shock-induced transcriptional activation of c-fos and zif-268 in hippocampus,xe2x80x9d J. Neurosci. Res., vol.37, pp. 54-61, (1994). PAF enhances glutamate release at the presynaptic level, which is antagonized by the plasma membrane-type receptor inhibitor BN-52021. See G. D. Clark et al., xe2x80x9cEnhancement of hippocampal excitatory synaptic transmission by platelet-activating factor,xe2x80x9d Neuron, vol.9, pp. 1211-1216 (1992); N. G. Bazan et al., xe2x80x9cPlatelet activating factor in the modulation of excitatory amino acid neurotransmitter release and of gene expression,xe2x80x9d J. Lipid Mediat. Cell Signal, vol. 14, pp.321-330 (1996a); and C. Chen et al., xe2x80x9cAttenuated long-term potentiation in hippocampal dentate gyrus neurons of mice deficient in the platelet-activating factor receptor,xe2x80x9d J. Neurophysiol., vol. 85, pp. 384-390 (2001).
PAF is also a retrograde messenger of long-term synaptic potentiation. See K. Kato et al., xe2x80x9cPlatelet activating factor as a potential retrograde messenger in Cal hippocampal long-term potentiation,xe2x80x9d Nature, vol. 367, pp. 175-179 (1994). Moreover, PAF is a transcriptional activator, and this action is blocked by the intracellular PAF antagonist LAU-8080 (BN-50730). See V. L. Marcheselli et al., 1994; V. L. Marcheselli et al., xe2x80x9cSustained induction of prostaglandin endoperoxide synthase-2 by seizures in hippocampus: Inhibition by a platelet-activating factor antagonist,xe2x80x9d J. Biol. Chem., vol. 271, pp. 24794-24799 (1996); Bazan et al., xe2x80x9cPlatelet-activating factor and retinoic acid synergistically activate the inducible prostaglandin synthase gene,xe2x80x9d Proc. Natl. Acad. Sci., vol. 91, pp. 5252-5256 (1994); P. K. Mukherjee et al., xe2x80x9cGlutamate receptor signaling interplay modulates stress-sensitive mitogen-activated protein kinases and neuronal cell death,xe2x80x9d J. Biol. Chem., vol.274, pp. 6493-6498 (1999).
Membrane-type PAF receptor antagonists exert dose-dependent protective effects in brain ischemia/reperfusion animal models. See Panetta et al., 1987; Nishida et al., 1996. The bioactive lipid PAF is a key modulator in glutamate signaling. Bazan et al., 1996a, b). The signaling of glutamate initiates a critical pathway during stroke that leads to activation of specific phospholipases A2, which in turn leads to more PAF production and activation of stress-sensitive protein kinases. This in turn leads to activation and translocation of transcription factors and hypoxia-inducible factors, followed by gene expression activation, in particular of the cyclooxygenase-2 (COX-2) gene. See Bazan et al., 1994; Marcheselli et al, 1994; and Marcheselli et al, 1996.
We have discovered a series of novel derivatives of 2,4,6-trimethyl-1,4-dihydropyridine-3,5-dicarboxylic acid (hereinafter, called the xe2x80x9cLAU-0900 series compoundsxe2x80x9d and each compound given an unique LAU-0900 number) and a novel synthesis of the derivatives. Surprisingly, by modifying the substituent of the 3-carboxylic acid group, new compounds were produced with higher activity as PAF receptor antagonists than commercially available PAF antagonists, e.g., WEB 2086BS, CV-6209, CV-3988, and the Ginkolide-B (BN-52021). In particular, these new compounds displayed a higher specificity for the intracellular binding site, and the duration of their effect was longer. These compounds were shown to protect neurons from brain damage that occurs in response to stroke and other cerebrovascular diseases, as ascertained both in a curative experimental design in a mouse model of stroke and in a preventive experimental design. These compounds are also effective in protecting damage caused by traumatic head injury and vasogenic edema. Moreover, these compounds were found to be nontoxic and cytoprotective of cells undergoing oxidative stress that would normally trigger apoptotic cell death; and to have activity as (a) antagonists of an intracellular platelet activating factor (xe2x80x9cPAFxe2x80x9d)-binding site, (b) inhibitors of PAF- and cytokine-mediated c-aminoterminal jun kinase (JNK) and extracellular regulated kinase (ERK), and (c) transcriptional inhibitors of COX-2 expression. It is believed that these compounds would be neuroprotective in other neural injuries, including spinal cord injury, status epilepticus-induced brain injury, traumatic head injury, neurodegenerative diseases (e.g., Alzheimer""s disease), ischemia-reperfusion injury to other organs (e.g., heart or kidney), ischemic retinal diseases, retinal degenerative diseases, and vasogenic injuries.